Nitrogen (N) and phosphorus (P) are the most limiting factors for plant growth. Some microorganisms improve the uptake and availability of N and P minimizing chemical fertilizers dependence.
Compared with the other major nutrients, such as nitrogen, phosphorus (P) is by far the least mobile and available to plants in most soil conditions. Although P is abundant in soils in both organic and inorganic forms, it is frequently a major or even the prime limiting factor for plant growth. Many soils throughout the world are P-deficient, because the free concentration (the form available to the plant), even in fertile soils, is generally low due to high reactivity of soluble P with calcium, iron, or aluminium that leads to P precipitation (36, 41). In addition, in developing countries chemical fertilizers, which provide the three major plant nutrients (N, P and potassium) are not widely used due to cost limitations. In these areas the direct application of ground Phosphate Rock (PR) is increasingly used, even if the P released from PR is often too low for crop growth (9, 38). It is known that many microorganisms, in particular of the genera Pseudomonas, Bacillus and Rhizobium, have the ability to change their metabolism in response to the phosphorus available for cellular growth. The switch in metabolism is mediated through the repression and induction of various genes whose products are involved in processes ranging from the uptake and acquisition of P sources to de novo synthesis of new cellular components (36, 18). Furthermore, in vitro studies showed that for some of these bacteria both the P-solubilizing activity and the production of the auxin indole-3-acetic acid (IAA) (39, 17) were observed, despite a direct correlation linking IAA production to P-solubilization was not demonstrated.
P uptake has been investigated in various microorganisms. Many bacteria, including S. meliloti, have at least two P transport systems, consistent with the high- and low-affinity transport systems. The high-affinity system is encoded by the phoCDET operon, and the low-affinity system is encoded by pit (in the orfA-pit operon). In S. meliloti the expression of genes encoding for both P transport systems is controlled by the PhoB activator. Under P-excess conditions, PhoB is inactive, and the phoCDET are not expressed. Under P-limiting conditions, the low-affinity Pit permease system is repressed by activated PhoB, while the high-affinity PhoCDET system is induced and becomes the primary mechanism of P transport (10). Many bacterial strains contain products pstSCAB homologs that function as high-affinity phosphate transporters. For S. meliloti 1021 a 1-bp deletion in the pstC ORF is probably responsible (via PhoB) for the moderate constitutive activation of 12 phosphate-starvation inducible genes, observed in the absence of phosphate stress (24, 43). In both plants and microorganisms, the primary mechanisms of PR solubilization are H+ excretion, organic acids production and acid phosphatase biosynthesis (2, 3). Organic acids, including acetate, lactate, malate, oxalate, succinate, citrate, gluconate, ketogluconate, etc. can form complexes with the iron or aluminum in ferric and aluminum phosphates, thus releasing plant-available phosphate into the soil (18, 22). Organic acids may also increase P availability by blocking P absorption sites on soil particles or by forming complexes with cations on soil mineral surface (36).
Mineralization of most organic phosphorus compounds is carried out by means of phosphatase enzymes. The major source of these enzymes in soil is considered to be of microbial origin. In particular, phosphatase activity is substantially increased in the rhizosphere. The pH of most soils ranges from acids to neutral values. Thus, acid phosphatases should play the major role in this process (36).
In the present invention, the P-solubilizing ability of a S. meliloti 1021 strain, RD64, and its effect on the growth of Medicago host plant were analysed.
The author used the S.meliloti-M. truncatula system since the microarrays were available for the bacterium and Medicago is a well recognized model system for indeterminate nodule development.
The RD64 strain has been previously engineered to overproduce IAA (11, 35), showing that it is able to release into liquid growth media up to 78-fold more IAA compared to wild type 1021 (12, 21). It was also previously reported that, as found for IAA-treated E. coli cells (7), RD64 is more resistant to salinity and other abiotic stresses (5). Medicago plants nodulated by this strain have a higher degree of protection against oxidative damage induced by salt stress (5). Furthermore, it was previously shown that IAA triggers induction of tricarboxylic acid cycle or citric acid cycle, TCA cycle enzymes in quite distant systems such as transformed human cells (15), E. coli (8) and S. meliloti (21) with a mechanism not yet understood.
To evaluate the global effects triggered by IAA overproduction in S. meliloti RD64, the gene expression pattern of wild type 1021 was compared with that of RD64 and 1021 treated with IAA and other four chemically or functionally related molecules by microarray analysis.
Among the genes differentially expressed in RD64 and IAA-treated 1021 cells, the author found two genes of pho operon. This unexpected finding led them to examine the mechanisms for mineral P solubilization in RD64 and the potential ability of this strain to improve Medicago growth under P-starved conditions. P-starved conditions are defined when bacteria, either 1021 or RD64, grow in media containing 1.0 mM K-phosphate. An increase in acid phosphatase activity and organic acids excretion was observed for RD64 strain in free-living conditions. Furthermore, the amount of organic acids exuded from the roots of Medicago plants nodulated by this strain was higher than that measured for plants nodulated by the 1021 wild type strain. This effect was connected to the enhanced P solubilization and plant dry weight production observed for these plants.